1. Field of the Invention
This invention relates generally to methods for detecting abusive use or stimulation of glucocorticoids in domestic animals, particularly in the context of the medical/legal issues associated with the performance of certain animals, e.g., race horses, racing greyhounds, sled dogs, etc., used in sporting contests. More specifically, this invention relates to methods for detecting use of supraphysiological levels of glucocorticoids by chemical analysis directed at alteration in animal liver glycogen metabolism function. Central to any chemical analysis is an understanding of the terms glycogen, glycogenesis, glycogenolysis and glucocorticoids.
Glycogen
Glycogen is the chief storage form of carbohydrate in animals and is analogous to starch in plants. The principle organ in which glycogen is stored in the body is the liver. The process of glycogen synthesis (glycogenesis), and that of its breakdown (glycogenolysis) is known to proceed by two separate pathways.
Glycogenesis
The initial reaction required for the entrance of glucose into the series of metabolic reactions which culminate in the synthesis of glycogen is phosphorylation of glucose at the C-6 position. Glucose is phosphorylated by adenosine triphosphate (ATP) in the liver by an irreversible enzymatic reaction which is catalyzed by a specific glucokinase. This undirectional phosphorylation permits the accumulation of glucose in the liver cell since the phosphorylated sugars do not pass freely in and out of the cell in contrast to the readily diffusable free sugars. The trapped glucose-6-phosphatase is converted to glucose-1-phosphatase, a reaction catalyzed by phosphoglucomutase. Glycogen is synthesized from the glucose-1-phosphate through reactions involving the formation of uridine derivatives. In the presence of polysaccharide primers and the enzyme glycogen synthetase, the glucose moiety of the urine derivatives is linked to the polysaccharide. Through repeated transfers of glucose, the polysaccharide chain is eventually lengthened until a glycogen molecule is formed.
Glycogenolysis
The breakdown of liver glycogen to glucose takes place by a second pathway. In the presence of inorganic phosphate, the glucose linkage of glycogen is successfully broken by active phosphorylases. Epinephrine and glucagon influence the phosphorolytic breakdown of glycogen to glucose. The phosphorolytic enzyme exists in the liver in two forms: an active form designated liver phosphorylase (LP) which contains phosphate and an inactive form designated dephosphorylase (dephospho-P), in which phosphate has been removed. The transformation between the active and the inactive forms are catalyzed by specific kinase enzymes. Normally the level of LP is low and the epinephrine and glucagon shifts the equilibrium toward a higher level of LP. The net result is an increased phosphorolytic breakdown of glycogen to glucose. A hyperglycemia is observed clinically following the injection of either of these two hormones.
Glucocorticoids
Glucocorticoids promote liver glycogen storage. This increase in liver glycogen storage has been attributed to glucocorticoid enhancement of gluconeogenesis, hyperglycemia, decreased glycogenolysis and decreased glucose oxidation.
Glucagon
Glucagon has been used in certain diagnostic procedures as well as in various pharmaceutical treatments. It is a polypeptide secreted by the alpha cells on the pancreas. The primary structure of porcine, bovine and human glucagon are identical. Glucagon is produced as a by-product of insulin production from pork and beef pancreases. Injections of glucagon are known to elevate blood glucose levels by causing hepatic glycogenolysis. Furthermore, it is known that under standardized conditions, glucagon induces reproducable hyperglycemia in test animals.
However, despite the knowledge of glycogenesis and glycogenolysis, it has not been heretofore fully appreciated that the intravenous administration of glucagon (glucagon tolerance test) may be used to detect the excessive storage of liver glycogen associated with supraphysiologic levels of glucocorticoids.
2. Prior Art
Injection tests have been used in the dog to differentiate pancreatic tumorbearing dogs in which insulin release is stimulated by the transient hyperglycemia product following glucagon administration; blood glucagon levels are then measured. (See Johnson RK: Insulinoma in the dog. Vet Clin North Am 7(3):629-635, 1977). The cat has also been used as an in vivo means of assaying small quantities of glucagon. (See Behrens OK, Broner W.: Glucagon, Vitamin, and Hormone, vol XVI-16:263-301, Academic Press, New York 1958.
Moreover, when used properly, glucocorticoids can be beneficial in alleviating inflammation before excessive tissue damage occurs. However, glucocorticoids do have the potential to be abused. Numerous reports document rapid suppression of the Hypothalamic-Hypophysis-Adrenocortical (HHA) axis after parenteral corticosteroid use. Along with injection, oral administration topical therapy has also been incriminated in suppression of and hypothalamic release of corticotropin-releasing factor and hypophysis ACTH, thus producing secondary adrenal atrophy. In addition to the detrimental effects of glucocorticoids on the HHA axis, they are also associated with a wide variety of detrimental effects such as delayed wound healing, infection, protein catabolism, steroid arthropathies and other Cushingoid conditions. The ability of exogenous glucocorticoids (or endogenous glucocorticoids produced by nonphysiological ACTH stimulation) to mask pain associated with inflammation is also well known. Intravenous and intramuscular injections of these substances, as well as their direct injection into the joints of race animals, are illegal, but are not uncommon practices. Consequently, the need for a simple and accurate test to detect evidence of such abuses clearly exists. This need is presently being met by various hematologic (neutrophilia, neutropenia, eosinopenia), hormonal (depressed ACTH response) and biochemical (serum, alkaline, phosphatase increases) tests which are, to varying degrees, capable of qualitative detection of glucocorticoid administration. However, none of these tests are sufficiently pronounced or consistent enough to be regarded as reliable indicators of exogenous administration of glucocorticoids and/or nonphysiologic ACTH stimulation of elevated glucocorticoid levels. Moreover, they are not well suited to field use. Liver biopsy for detection of steroid hepatopathy is a sensitive and consistent test; but it is generally regarded as being too risky and impractical in most performance animal test situations. Consequently, the most widespread method currently used for detecting use of exogenous glucocorticoids and ACTH stimulation depends upon detection of the propylene glycol base often used in many glucocorticoid or ACTH formulations. However, this technique is totally ineffective in detecting aqueous preparations of those glucocorticoids which are frequently injected into the joints of race animals. Hence complete definitions of the role of glucocorticoids in race animal sporting events has been hampered by the lack of a specific and sensitive assay for glucocorticoids. Without a satisfactory technique to determine the presence of glucocorticoids in readily available biologic substances such as the animal's blood, any accusations or conclusions regarding the possible abuse of glucocorticoids must remain presumptive or tentative. In order to obviate this problem, Applicant has developed a safe, reliable test for detecting evidence of supraphysiologic glucocorticoid levels, regardless of the chemical nature of the base or carrier in which the glucocorticoid is administered. This test also has other diagnostic aspects which are covered in, Roberts, Stevens M., et al, Effect of Opthalmic Prednisolone Acetate on the Canine Adrenal Gland and Hepatic Function, Am. J. Vet. Res., Vol. 45, No. 9 (September 1984) which is specifically incorporated herein by reference.